Monitoring electrical activity in neurons is of critical importance for understanding the dynamic manner in which brain cells communicate. Currently, electrophysiology can provide exquisitely precise measurements of voltage changes and action potentials in a single cell, but the spatial information derived from electrophysiological measurement is limited. Optical imaging provides an attractive solution for monitoring voltage changes in multiple neurons simultaneously. Unfortunately, no single optical probe for voltage has been able to supply the requisite characteristics of a probe suitable for sensitive detection of action potentials and other sub-threshold events. In this research, a method is devised for detecting changes in voltage in neuronal cells which incorporates large fluorescence changes (1-2%/mV), fast kinetics (submicrosecond), negligible capacitative load on the cell, genetic targetability, and synthetic tractability. The efficiency of photo- induced electron transfer (PeT) between an aniline donor and a xanthene-based fluorophore acceptor will be altered in the presence of an applied electric field. The magnitude of the change will be proportional to the strength of the field and the length of the dipole formed upon excitation to the singlet excited state. Given that the electric field in a neuronal context will be relatively constant at approximately 105 V/cm, large changes in fluorescence upon application of an electric field can be realized by increasing the dipole, or the distance from donor to acceptor. To combat the exponential distance dependence of electron transfer, which will significantly impair the ability of a PeT-based sensor to respond to voltage, a molecular wire will be used to connect donor to acceptor. Molecular wires decrease the distance dependence of electron transfer, by changing the mechanism of electron transfer from superexchange at close distances to electron hopping at very large distances. A molecular wire will allow effective electron transfer while maximizing the change in fluorescence upon introduction of an electric field. The sensor can be further improved by targeting the probe, through the use of a chemically reactive handle, to genetically defined neuronal sub-populations. Expression of membrane- bound fusion proteins that covalently self-ligate orthogonal chemical handles to themselves will allow for targeting of the sensor to specific sub-types of neurons, increasing the signal-to-noise ratios of the probe in response to voltage changes and allowing for investigation of genetic sub-types of neurons within heterogeneous samples. Development of a fluorescent probe for voltage which can deliver large fluorescence increases in response to changes in voltage while maintaining good temporal fidelity and avoiding capacitative loading will be of broad interest for studying a wide range of neurological systems. PUBLIC HEALTH RELEVANCE: Optical imaging of changes in electrical potential in neuronal cells offers an attractive method for studying the dynamics of neuronal communication. Traditional optical methods for monitoring neuronal activity suffer from low signal-to-noise ratios in response to changes in voltage across the cellular membrane. This research will develop new molecular wire-based fluorescent probes for optically monitoring voltage in living cells with high spatial and temporal resolution.